Method for using hydratase or a hydratase-amidase fusion for stereospecifically bioconverting certain racemic nitriles to the corresponding enatiomeric R--or S-amide or s-carboxylic acid

ABSTRACT

The present invention provides a nitrile hydratase nucleic acid fragment isolated from Pseudomonas putida which encodes a nitrile hydratase activity capable of catalyzing the hydrolysis of certain racemic nitrites to the corresponding R- or S-amides. Also provided are transformed microorganisms capable of the active expression of said nitrile hydratase activity. Additionally, the invention provides a transformant harboring the nitrile hydratase gene in conjunction with an amidase gene, both of which may be co-expressed producing active nitrile hydratase and amidase enzymes respectively. Methods for the production of such enantiomeric materials are also provided.

This is a division of application Ser. No. 08/726,136 filed Oct. 4,1996, now U.S. Pat. No. 5,811,286, claiming benefit of P1 case60/004,914 filed Oct. 6, 1995.

FIELD OF INVENTION

The present invention relates to the field of molecular biology andmethods for the isolation and expression of foreign genes in recombinantmicroorganisms. More specifically, the invention relates to theisolation, sequencing, and recombinant expression of nucleic acidfragments (genes) encoding a stereospecific, nitrile hydratase (NHase)activity capable of catalyzing the hydrolysis of certain racemicnitrites to the corresponding R- or S- amides. Additionally, theinvention relates to the co-expression of the nitrile hydratase nucleicacid fragment with a nucleic acid fragment encoding a stereospecificamidase activity capable of converting a racemic mixture of R- and S-amides to the corresponding enantiomeric R- or S- carboxylic acids.

BACKGROUND

Many agrochemicals and pharmaceuticals of the general formulaX--CHR--COOH are currently marketed as racemic or diastereomer mixtures.In many cases the physiological effect derives from only oneenantiomer/diastereomer where the other enantiomer/diastereomer isinactive or even harmful. Methods for synthesizing enantiomers arebecoming increasingly important tools for the production of chemicals ofenantiomer purity. To date, however, no recombinant, stereospecificNHase has been described capable of catalyzing the hydrolysis of certainracemic nitrites to the corresponding R- or S- amides.

Methods for the selective preparation of stereospecific amides fromnitrites are known and incorporate microorganisms possessing nitrilehydratase activity (NHase). These NHases catalyze the addition of onemolecule of water to the nitrile, resulting in the formation of theamide free product according to Reaction 1:

Reaction 1

    R--CN+H.sub.2 O→RCONH.sub.2

Similarly, methods for the stereospecific production of carboxylic acidsare known and incorporate microorganisms possessing an amidase (Am)activity. In general amidases convert the amide product of Reaction 1 tothe acid free product plus ammonia according to Reaction 2:

Reaction 2

    RCONH.sub.2 →RCOOH+NH.sub.3

A wide variety of bacterial genera are known to possess a diversespectrum of nitrile hydratase and amidase activities includingRhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus,Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus. Forexample, nitrile hydratase enzymes have been isolated from Pseudomonaschlororaphis, B23 Nishiyama, M. J., Bacteriol., 173:2465-2472 (1991)!Rhodococcus rhodochrous J1 Kobayashi, M., Biochem. Biophys. Acta,1129:23-33 (1991)! Brevibacterium sp. 312(Mayaux et al., J. Bacteriol.,172:6764-6773 (1990)), and Rhodococcus sp. N-774 Ikehata, O., Nishiyama,M., Horinouchi, S., Beppu, T., Eur. J. Biochem., 181: 563-570(1989)!. Nodisclosure of any stereoselective activity is made for any of theseenzymes. Only two disclosures have been made for stereoselective nitrilehydratase activity in native bacterial strains. The Applicants havedisclosed a stereospecific nitrile hydratase from P. putida NRRL-18668WO 92/05275 (1990)!.

Wildtype microorganisms known to possess nitrile hydratase activity havebeen used to convert nitrites to amides and carboxylic acids. Forexample, EPA 326,482 discloses the stereospecific preparation ofaryl-2-alkanoic acids such as 2-(4-chlorophenyl)-3-methylbutyric acid bymicrobial hydrolysis of the corresponding racemic amide using members ofBrevibacterium and Corynebacterium. Similarly, U.S. Pat. No. 4,366,250teaches the use of Bacillus, Bacteridium, Micrococcus and Brevibacteriumin a method for the preparation of L-amino acids from the correspondingracemic amino nitriles. Finally, WO 92/05275 teaches abiologically-catalyzed method for converting a racemic alkyl nitrile tothe corresponding R- or S-alkanoic acid through an intermediate amideusing members of the bacterial genera Pseudomonas spp. (e.g., putida,aureofaciens, Moraxella spp.) and Serratia (e.g., Serratialiquefaciens).

In addition to the use of wildtype organisms, recombinant organismscontaining heterologous genes for the expression of nitrile hydrataseare also known for the conversion of nitrites. For example, Cerebelaudet al., (WO 9504828) teach the isolation and expression in E. coli ofnitrile hydratase genes isolated from C. testosteroni. The transformedhosts effectively convert nitrites to amides where the nitrile substrateconsists of one nitrile and one carboxylate group. However, WO 9504828does not teach a stereospecific conversion of nitrites.

Similarly, Beppu et al., (EP 5024576) disclose plasmids carrying bothnitrile hydratase and amidase genes from Rhodococcus capable oftransforming E. coli where the transformed host is then able to useisobutyronitrile and isobutyroamide as enzymatic substrates. However, EP5024576 does not teach a stereospecific conversion of nitrites oramides.

As with nitrile hydratases, microorganisms possessing amidase activityhave been used to convert amides to carboxylic acids. In U.S. Ser. No.08/403911, Applicants disclose a method for converting an (S)-amide, orstereospecifically converting a mixture of (R)- and (S)-amides to thecorresponding enantiomeric (S)-carboxylic acid by contacting said amidewith Pseudomonas chlororaphis B23 in a solvent. This method uses awildtype microorganism and does not anticipate a recombinant catalyst orheterologous gene expression. Blakey et al., FEMS Microbiology Letters,129:57-62 (1995) disclose a Rhodococcus sp. having activity against abroad range of nitrites and dinitriles and able to catalyzeregio-specific and stereo-specific nitrile biotransformations.

Genes encoding amidase activity have been cloned, sequenced, andexpressed in recombinant organisms. For example, Azza et al., (FEMSMicrobiol. Lett. 122, 129, (1994)) disclose the cloning andover-expression in E. coli of an amidase gene from Brevibacterium sp.R312 under the control of the native promoter. Similarly, Kobayashi etal., (Eur. J. Biochem., 217, 327, (1993)) teach the cloning of both anitrile hydratase and amidase gene from R. rhodococcus J1 and theirco-expression in E. coli.

What is needed and inventive over the prior art is a method for thestereospecific conversion of racemic alkyl nitriles to the correspondingR- or S-alkanoic acids using a recombinant organism.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid fragments encoding:

1) the α subunit of a stereospecific nitrile hydratase enzyme, said genehaving at least a 64% base homology with the α subunit coding region ofthe Rhodococcus rhodochrous J1 L-NHase gene Kobayashi, M., Biochem.Biophys. Acta, 1129:23-33 (1991)! and said enzyme capable of catalyzingthe hydrolysis of racemic aryl-2-alkane nitrites to the corresponding R-or S- amides; and

2) the β subunit of a stereospecific nitrile hydratase enzyme, said genehaving at least a 52% base homology with the β subunit coding region ofthe Rhodococcus rhodochrous J1 L-NHase gene and said enzyme capable ofcatalyzing the hydrolysis of racemic aryl-2-alkane nitriles to thecorresponding R- or S-amides.

Another embodiment of the invention is a nucleic acid fragmentcomprising the nucleic acid fragments encoding both the α and β subunitsof a stereospecific nitrile hydratase enzyme described above, saidenzyme capable of catalyzing the hydrolysis of racemic aryl-2-alkanenitrites to the corresponding R- or S-amides.

A further embodiment of the invention is a nucleic acid fragmentencoding the α subunit of a stereospecific nitrile hydratase enzyme,said nucleic acid fragment having the nucleotide sequence as representedin SEQ ID NO.:3 and said enzyme capable of catalyzing the hydrolysis ofracemic alkyl nitrites to the corresponding R- or S- amides.

A further embodiment of the invention is a nucleic acid fragmentencoding the β subunit of a stereospecific nitrile hydratase enzyme,said nucleic acid fragment having the nucleotide sequence as representedin SEQ ID NO.:4 and said enzyme capable of catalyzing the hydrolysis ofracemic alkyl nitriles to the corresponding R- or S- amides.

Still another embodiment of the invention is a nucleic acid fragmentencoding both the α and β subunits of a stereospecific nitrile hydrataseenzyme, said nucleic acid fragment having the nucleotide sequence asrepresented in SEQ ID NO.:17 and said enzyme capable of catalyzing thehydrolysis of racemic aryl-2-alkane nitrites to the corresponding R- orS- amides.

Further embodiments of the invention include

1) the polypeptide a subunit of a stereospecific nitrile hydrataseenzyme, said α subunit having the amino acid sequence as represented inSEQ ID NO.:1 and said enzyme being capable of catalyzing the hydrolysisof racemic aryl-2-alkane nitrites to the corresponding R- or S- amides;and

2) the polypeptide β subunit of a stereospecific nitrile hydrataseenzyme, said β subunit having the amino acid sequence as represented inSEQ ID NO.:2 and said enzyme being capable of catalyzing the hydrolysisof racemic aryl-2-alkane nitriles to the corresponding R- or S- amides.

A further embodiment of the invention is a stereospecific nitrilehydratase enzyme, said enzyme comprising the combined α and β subunitshaving the respective amino acid sequences SEQ ID NOs.:1 and 2 in properconformation such that said enzyme catalyzes the hydrolysis of racemicaryl-2-alkane nitriles to the corresponding R- or S- amides.

A still further embodiment of the invention is a 6.5 kb nucleic acidfragment encoding a nitrile hydratase enzyme and the accessory nucleicacid fragments necessary for the enzymes's active expression and furthercharacterized by the restriction fragment map shown in FIG. 2. This 6.5kb nucleic acid fragment is incorporated into an expression vectorcapable of transforming a suitable host cell for the expression ofactive stereospecific nitrile hydratase as characterized by the plasmidmap shown in FIG. 3.

The invention further provides a region of the P. putidia genomeencompassed within the 6.5 kb fragment, designated P14K, which encodes apolypeptide that is necessary for the bioactivity of the stereospecificnitrile hydratase enzyme isolated from Pseudomonas putida NRRL-18668.

Additionally the invention provides a nucleic acid fragment encoding a18668 amidase having an amino acid sequence as represented in SEQ IDNO.:28, wherein the amino acid sequence may encompass amino acidsubstitutions, deletions or additions that do not alter the function ofsaid amidase. The 18668 amidase is isolated from Pseudomonas putidaNRRL-18668 and is distinct from the amidase isolated from Pseudomonaschlororaphis B-23 (FERM B-187).

The present invention further provides recombinant hosts, transformedwith the nucleic acid fragment encoding a 18668 amidase and/or the genesencoding the α, β nitrile hydratase subunits and the P14K region of thePseudomonas putida NRRL-18668 genome.

The invention also provides methods for the conversion of racemicnitrites to the corresponding R- or S-amides or correspondingenantiomeric R- or S-carboxylic acids using the above transformed hostscontaining nucleic acid fragments encoding a 18668 amidase and/or thegenes encoding the α, β nitrile hydratase subunits and the P14K regionof the Pseudomonas putida NRRL-18668 genome.

Other embodiments of the invention are:

1) a transformed microbial host cell comprising the nucleic acidfragment represented by SEQ ID NO.:17 wherein said host cell expressesactive nitrile hydratase enzyme capable of catalyzing the hydrolysis ofracemic aryl-2 alkane nitriles to the corresponding R- or S- amides; and

2) a transformed microbial host cell comprising the 6.5 kb nucleic acidfragment characterized by the restriction map shown in FIG. 2 whereinsaid host cell expresses active nitrile hydratase enzyme capable ofcatalyzing the hydrolysis of racemic aryl-2 alkane nitriles to thecorresponding R- or S- amides.

Other embodiments of the invention are host cells transformed withnucleic acid fragments represented by SEQ ID NO.:17 or the restrictionmaps of FIGS. 2 and 3, wherein the host cell is selected from the groupconsisting of bacteria of the genera Escherichia, Pseudomonas,Rhodococcus, Acinetobacter, Bacillus, and Streptomyces, yeast of thegenera Pichia, Hansenula, and Saccharomyces, and filamentous fungi ofthe genera Aspergillus, Neurospora, and Penicillium.

A particular embodiment of the invention is Escherichia coli transformedwith the nucleic acid fragment represented by SEQ ID NO.:17 or thenucleic acid fragment represented by the restriction map of FIG. 2.

A further embodiment of the invention is an expression vector describedin FIG. 6 comprising 1) a 5.0 kb nucleic acid fragment from the 6.5 kbfragment of claim 10, and 2) a nucleic acid fragment having the nucleicacid sequence as given in SEQ ID NO.:20, wherein said nucleic acidfragment encodes an amidase enzyme, and wherein said expression vectoris capable of transforming suitable host cells for the co-expression ofactive stereospecific nitrile hydratase and amidase. A furtherembodiment is a host cell transformed with this expression vectorwherein more particularly the host is selected from the group consistingof the genera Escherichia, Pseudomonas, Rhodococcus, Acinetobacter,Bacillus, Streptomyces, Hansenula, Saccharomyces, Pichia, Aspergillus,Neurospora, and Penicillium. A further embodiment is Escherichia coliSW17 transformed with pSW17.

A further embodiment of the invention is a method for converting anitrile of the formula ##STR1## wherein: A is selected from the groupconsisting of: ##STR2## to the corresponding amide comprising contactingsaid nitrile with the transformed host cell containing a nucleic acidfragment having the nucleotide sequence represented by SEQ ID NO.:17that stereospecifically converts the racemic nitrile to thecorresponding enantiomeric R- or S-amide, the host cell selected fromthe group consisting of Escherichia, Pseudomonas, Rhodococcus,Acinetobacter, Bacillus, Streptomyces, Hansenula, Saccharomyces, Pichia,Aspergillus, Neurospora, and Penicillium.

The Applicants also provide a method for the conversion of the abovedescribed nitrile to corresponding enantiomeric (R) or (S)-carboxylicacid by contacting the nitrile with the transformed host comprising anexpression vector comprising a nucleic acid fragment represented by FIG.2 and the nucleic acid sequence of SEQ ID NO.:20, the host cell selectedfrom the group consisting of Escherichia, Pseudomonas, Rhodococcus,Acinetobacter, Bacillus, Streptomyces, Hansenula, Saccharomyces, Pichia,Aspergillus, Neurospora, and Penicillium.

A further embodiment of the invention is a nucleic acid fragmentencoding the α and β subunits of a stereospecific nitrile hydrataseenzyme, said portion of the nucleic acid fragment encoding the α subunithaving at least a 64% base homology to the Rhodochrous J1 L-NHase geneand said portion of the nucleic acid fragment encoding the β subunithaving a 52% base homology to the Rhodochrous J1 L-NHase gene, and saidenzyme capable of catalyzing the hydrolysis of racemic aryl-2-alkanenitrites to the corresponding R- or S- amides.

Yet another embodiment of the invention is the polypeptide encoded byany one of the nucleic acid fragments of the invention.

Embodiments of the invention are plasmids pSW2 carried in SW2 anddesignated as ATCC 69888, pSW17 carried in SW17 and designated as ATCC69887, pSW50 carried in P. pastoris SW50.2 and designated as ATCC 74391,pSW37 carried in E. coli SW37 and designated as ATCC 98174, and pSW23carried in E. coli SW23 and designated as ATCC 98175.

BRIEF DESCRIPTION OF THE FIGURES BIOLOGICAL DEPOSITS AND SEOUENCELISTING

FIG. 1 is a plasmid map of the plasmid pSW1 containing a 6.5 kb DNAfragment which encodes the α and β subunits of the nitrile hydrataseenzyme isolated from P. putida (NRRL-18668).

FIG. 2 is a restriction map of the 6.5 kb nucleic acid fragment whichincludes the nitrile hydratase gene isolated from P. putida (NRRL-18668)showing the location of the α and β subunits.

FIG. 3 is a plasmid map of the plasmid pSW2 created by inserting the 6.5kb DNA fragment comprising the genes encoding the α and β subunits ofnitrile hydratase into the wide-host-range vector pMMB207.

FIG. 4 is a plasmid map of the plasmid pSW5 created by inserting a 2.8kb subclone of the 6.5 kb nucleic acid fragment comprising the genesencoding the α and β subunits of nitrile hydratase into thewide-host-range vector pMMB207.

FIG. 5 is a western blot analysis showing the production of NRRL-18668nitrile hydratase protein in E. coli. (A) Coomassie Blue stainedSDS-PAGE gel of protein extracts from uninduced (u) and induced (i) E.coli transformed with the plasmid pSW2. (B) Western blot analysis ofduplicate gel shown in (A) using anti-NH sera. M, protein molecularweight markers; NH, nitrile hydratase protein from NRRL-18668. Arrowindicates NH.

FIG. 6 is a plasmid map of the plasmid pSW17 created by inserting a 1.5kb DNA fragment comprising the gene encoding amidase from Pseudomonaschlororaphis B23, and a 5.0 kb subclone of the 6.5 kb DNA fragmentcomprising the genes encoding the α and β subunits of nitrile hydrataseinto the wide-host-range vector pMMB207.

FIG. 7 illustrates the nucleotide and amino acid sequences of thePseudomonas putida (NRRL-18668) α and β nitrile hydratase coding regionsalso found in SEQ ID NO.:17.

FIG. 8 is a restriction map of the 6.5 kb nucleic acid fragment whichincludes the nitrile hydratase gene isolated from P. putida (NRRL-18668)plus sequence upstream of the EcoR1 site (shown in FIG. 2) including anew Pst1 site.

FIG. 9 is a restriction map of the 6.5 kb nucleic acid fragment whichincludes the nitrile hydratase gene isolated from P. putida (NRRL-18668)plus sequence upstream of the new Pst1 site (shown in FIG. 8) includinga new EcoR1 site.

FIG. 10 is a restriction map of an 8 kb nucleic acid fragment showingthe 6.5 kb nucleic acid fragment which includes the nitrile hydratasegene isolated from P. putida (NRRL-18668), P14K, and the region encodinga P. putida (NRRL-18668) amidase enzyme.

FIG. 11 is a plasmid map of pHIL-D4B2 created by replacing the 0.9 kbEcoR1/Xba1 fragment in pHIL-D4 with the 0.9 kb EcoRl/Xbal fragment frompAO815.

FIG. 12 is a plasmid map of pSW46 created by the insertion of the α geneof the nitrile hydratase enzyme into the EcoR1 site of pHIL-D4B2.

FIG. 13 is a plasmid map of pSW47 created by the insertion of the β geneof the nitrile hydratase enzyme into the EcoR1 site of pHIL-D4B2.

FIG. 14 is a plasmid map of pSW48 created by the insertion of the P14Kgene into the EcoR1 site of pHIL-D4B2.

FIG. 15 is a plasmid map of pSW49 containing the α and β expressioncassettes from pSW46 and pSW47.

FIG. 16 is a plasmid map of pSW50 containing the α, β and P14Kexpression cassettes from pSW46, pSW47 and pSW48.

FIG. 17 is a plasmid map of pSW37 containing the expression cassette forthe amidase isolated from P. putida (NRRL-18668).

FIG. 18 is a plasmid map of pSW23 containing the expression cassette forthe amidase, α, β and P14K isolated from P. putida (NRRL-18668).

Applicants have provided sequence listings 1-28 in conformity with 37C.F.R. 1.821-1.825 and Appendices A and B ("Requirements for ApplicationDisclosures Containing Nucleotides and/or Amino Acid Sequences") and inconformity with "Rules for the Standard Representation of Nucleotide andAmino Acid Sequences in Patent Applications" and Annexes I and II to theDecision of the President of the EPO, published in Supplement No. 2 toOJ EPO, 12/1992.

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure:

    __________________________________________________________________________    Depositor Identification Reference                      Int'l. Depository Designation                                   Date of Deposit    __________________________________________________________________________    Pseudomonas Putida                      NRRL 18668   6 July 1990    Escherichia coli SW2 carrying pSW2                      ATCC 69888   15 August 1995    Escherichia coli SW17 carrying pSW17                      ATCC 69887   15 August 1995    Pichia pastoris SW50.2 carrying pSW50                      ATCC 74391   20 September 1996    E. coli SW37 carrying pSW37                      ATCC 98174   20 September 1996    E. coli SW23 carrying pSW23                      ATCC 98175   20 September 1996    __________________________________________________________________________

As used herein, "NRRL" refers to the Northern Regional ResearchLaboratory, Agricultural Research Service Culture CollectionInternational Depository Authority located at 11815 N. UniversityStreet, Peoria, Ill. 61604 U.S.A. The "NRRL No." is the accession numberto cultures on deposit at the NRRL.

As used herein, "ATCC" refers to the American Type Culture CollectionInternational Depository Authority located at 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. The "ATCC No." is the accession numberto cultures on deposit with the ATCC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides genes derived from Pseudomonas putida(NRRL-18668) which encode two polypeptides, which, in combination, havethe ability to act as a catalyst to selectively hydrate one nitrileenantiomer in a racemic mixture to produce the chiral amide. Thisinvention also provides a recombinant nucleic acid fragment containingthe genes and a set of transformed microbial cell hosts containing therecombinant nucleic acid fragment. The invention further provides amethod for the production of the polypeptide catalysts using thetransformed microbes and the use of the catalyst in chiral amideproduction. Additionally, the invention provides for the co-expressionin a transformed host of the nitrile hydratase genes with the genesencoding a stereospecific amidase derived from Pseudomonas chlororaphisB-23 (FERM B-187) for the production of chiral acids.

The following definitions are used herein and should be referred to forinterpretation of the claims and the specification.

Abbreviations:

CPIA--2-(4-chlorophenyl)-3-methylbutyric acid

CPIAm--2-(4-chlorophenyl)-3-methylbutyramide

CPIN--2-(4-chlorophenyl)-3-methylbutyronitrile

GC--Gas Chromatography

HPLC--High-Performance Liquid Chromatography

IPTG--isopropyl-b-D-thiogalatopyranoside

SDS Page--Sodium dodecyl sulfate polyacrylimide gel electrophoresis

The term "nitrile hydratase" refers to an enzyme isolated from thebacteria Pseudomonas putida (NRRL-18668) which is characterized by itsability to convert a racemic alkyl nitrile to the correspondingenantiomeric R- or S-amide through an intermediate amide where thestarting nitrile is: ##STR3## and wherein: A is selected from the groupconsisting of: ##STR4##

More specifically, the enzyme has an ability to connect the racemicalkyl nitrile to the corresponding enantiomeric R- or S-alkanoic acidthrough an intermediate amide.

The instant nitrile hydratase is further defined by the amino acidsequences of its α and β subunits as respectively given in SEQ ID NO.:1and SEQ ID NO.:2 which are encoded by the α and β nitrile hydratasesubunit genes whose base sequences are respectively given by SEQ IDNO.:3 and SEQ ID NO.:4.

The term "amidase" refers to an enzyme naturally found in the bacteriumPseudomonas putida B23(FERM B-187) which is characterized by its abilityto convert amides of the structure: ##STR5## wherein: A is selected fromthe group consisting of: ##STR6## R¹ is C₁ -C₄ alkyl; R² is H; F; Cl;Br; OH; C₁ -C₃ alkyl; OCF₂ H; or H₂ C═C(CH₃)CH₂ NH; and

R³ is H; F; Cl; Br; OH; C₁ -C₃ alkyl; or C₁ -C₃ alkoxy;

to the corresponding enantiomeric (R) or (S)-carboxylic acid. Theamidase of the instant invention is further identified by the amino acidsequence given in Nishiyama et al., Bacterial., 173:2465-2472 (1991) andthe DNA base sequence disclosed in SEQ ID NO.:20.

The term "118668 amidase" refers to an enzyme naturally found in thebacterium Pseudomonas putida NRRL-18668 which is characterized by itsability to convert C3 to C6 amides to the corresponding acids. Inaddition, as described in PCT/DK91/00189, the 18668 amidase ischaracterized by the ability to convert some (R,S )-aryl-2-alkanenitriles to the corresponding enantiomerically enriched (R) or(S)-carboxylic acid. The amidase of the instant invention is furtheridentified by the amino acid sequence given in SEQ ID NO.:28 and the DNAbase sequence disclosed in SEQ ID NO.:27. The "18668 amidasel" isdistinct from the amidase isolated from bacterium Pseudomonas putidaB23(FERM B-187).

The term "P14K gene" refers to a region of the Pseudomonas putidaNRRL-18668 genome encoding a polypeptide as given by SEQ ID NO.:22having the base sequence as given by SEQ ID NO.:21, where the expressionof the P14K gene is essential for the bioactivity of the Pseudomonasputida NRRL-18668 nitrile hydratase enzyme. The term "P14K polypeptide"(or "P14K protein") refers to the active polypeptide encoded by the P14Kregion.

"Transformation" refers to the acquisition of new genes in a cell by theincorporation of nucleic acid.

The term "nucleic acid" refers to complex compounds of high molecularweight occurring in living cells, the fundamental units of which arenucleotides linked together with phosphate bridges. Nucleic acids aresubdivided into two types: ribonucleic acid (RNA) and deoxyribonucleicacid (DNA).

The terms "host cell" and "host organism" refer to a microorganismcapable of incorporating foreign or heterologous genes and expressingthose genes to produce an active gene product.

The terms "foreign gene", "foreign DNA", "heterologous gene", and"heterologous DNA" refer to genetic material native to one organism thathas been placed within a host organism.

The terms "recombinant organism", "transformed host", and "transformedmicrobial host" refer to an organism having been transformed withheterologous or foreign genes. The recombinant organisms of the presentinvention express foreign genes encoding active nitrile hydratase andamidase enzymes.

The term "nucleic acid fragment" refers to a fragment of DNA that mayencode a gene and/or regulatory sequences preceding (5"non-coding) andfollowing (3"non-coding) the coding region (gene).

The term "expression" refers to the transcription and translation togene product from a gene coding for the sequence of the gene product,usually a protein.

The terms "plasmid" and "vector" refer to an extra chromosomal elementoften carrying genes which are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA molecules.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage sequences, linear or circular, of a single-or double-stranded DNA or RNA, derived from any source.

The term "cassette" refers to a number of nucleotide sequences whichhave been joined or recombined into a unique construction. An"expression cassette" is specifically comprised of a promoter fragment,a DNA sequence for a selected gene product, and a transcriptionaltermination sequence.

The terms "restriction endonuclease" and "restriction enzyme" refer toan enzyme which catalyzes hydrolytic cleavage within a specificnucleotide sequence in double-stranded DNA.

The term "promoter" refers to a sequence of DNA, usually upstream of (5'to) the protein coding sequence of a structural gene, which controls theexpression of the coding region by providing the recognition for RNApolymerase and/or other factors required for transcription to start atthe correct site.

A "fragment" constitutes a fraction of the complete nucleic acidsequence of a particular region. A fragment may constitute an entiregene.

The terms "peptide", "polypeptide" and "protein" are usedinterchangeably to refer to the gene product expressed.

The terms "encoding" and "coding" refer to the process by which a gene,through the mechanisms of transcription and translation, produces anamino acid sequence. The process of encoding a specific amino acidsequence includes DNA sequences that may involve base changes that donot cause a change in the encoded amino acid, or which involve basechanges which may alter one or more amino acids, but do not affect thefunctional properties of the protein encoded by the DNA sequence. It istherefore understood that the invention encompasses more than thespecific exemplary sequences. Modifications to the sequence, such asdeletions, insertions, or substitutions in the sequence which producesilent changes that do not substantially affect the functionalproperties of the resulting protein molecule are also contemplated. Forexample, alteration in the gene sequence which reflect the degeneracy ofthe genetic code, or which result in the production of a chemicallyequivalent amino acid at a given site, are contemplated. Thus, a codonfor the amino acid alanine, a hydrophobic amino acid, may be substitutedby a codon encoding another less hydrophobic residue, such as glycine,or a more hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a biologically equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the protein molecule would also not be expectedto alter the activity of the protein. In some cases, it may, in fact, bedesirable to make mutants of the sequence in order to study the effectof alteration on the biological activity of the protein. Each of theproposed modifications is well within the routine skill in the art, asis determination of retention of biological activity in the encodedproducts. Moreover, the skilled artisan recognizes that sequencesencompassed by this invention are also defined by their ability tohybridize, under stringent conditions (0.1× SSC, 0.1% SDS, 65° C.), withthe sequences exemplified herein.

"Homology" refers to the degree to which two nucleic acid fragmentscontain the same base sequence. "Homology" is determined by theoperation of an algorithim and is expressed as a percentage of the basesequence that is the same in both fragments.

Applicants have accomplished the following which are discussed in moredetail below and in the Examples:

I. identified and cloned genes for (i) a stereospecific NHase fromNRRL-18668, comprising both the α-subunit of the amino acid sequenceidentified in the Sequence Listing by SEQ ID NO.:1 and the β-subunit ofthe amino acid sequence identified in the Sequence Listing by SEQ IDNO.:2; (ii) an amidase from NRRL-18668 with deduced amino acid sequenceidentified in the Sequence Listing by SEQ ID NO.:28; (iii) a gene fromNRRL-18668 designated P14K which is essential for NRRL-18668 NHaseactivity and with deduced amino acid sequence identified in the SequenceListing by SEQ ID NO.:22;

II. obtained DNA sequences encoding the α-subunit identified in theSequence Listing by SEQ ID NO.:3; and the β-subunit identified in theSequence Listing by SEQ ID NO.:4; and the amidase enzyme identified inthe Sequence Listing by SEQ ID NO.:27; and the P14K polypeptideidentified in the Sequence Listing by SEQ ID NO.:21;

III. constructed recombinant DNA plasmids containing the genes asdescribed in I above located within an 8.0 kb DNA fragment as describedin FIG. 10.

IV. transformed microbial hosts with the plasmids described in III aboveas described in FIGS. 3, 15, and 16;

V. developed a method for the production of stereospecific NHase whichcomprises growing a transformed host described in IV and recovering thenitrile hydrating activity from the culture;

VI. developed a method for the production of chiral amides whichcomprises stereospecifically hydrating the nitrile using the nitrilehydrating activity recovered in V;

VII. developed a method for the production of chiral amides whichcomprises stereospecifically hydrating the nitrile using the nitrilehydrating activity recovered in V for the production of chiral amidesusing isolated microbial cells as described in IV, the treated matterthereof, or a fixed form of them;

VIII. constructed recombinant DNA plasmids containing the NHase genes asdescribed in I above, in combination with the amidase gene derived fromPseudomonas chlororaphis B23 (FERM B-187) or the amidase gene describedin I above;

IX. transformed microbial hosts with the plasmids described in VIIIabove as described in FIGS. 6 and 18;

X. developed a method for the production of NHase and amidase whichcomprises growing a transformed host described in IX and recovering thenitrile hydrating and amide hydrating activity from the culture; and

XI. developed a method for the production of chiral amides and chiralacids which comprises stereoselective hydration of the nitrile and itsamide products using the NHase and amidase activities recovered in V forthe production of the chiral products using isolated microbial cells asdescribed in IX, the treated matter thereof, or a fixed form of them toproduce chiral products.

I. ISOLATION AND CLONING OF THE NITRILE HYDRATASE GENE

A. Isolation and Partial Amino Acid Sequencing of the Nitrile HydrataseEnzyme:

The instant invention provides a nitrile hydratase enzyme which isdefined above. The nitrile hydratase of the present invention wasisolated and purified from Pseudomonas putida (NRRL-18668). Bacterialnitrile hydratases are known to be generally comprised of structurallydistinct α and β subunits (Hashimoto et al., Biosci., Biotechnol.,Biochem., 58(10), 1859-65 (1994)). The instant nitrile hydratase wasseparated into α and β subunits using HPLC methodology. Methods for thepurification and separation of enzymes by HPLC are common and known inthe art. See, for example, Rudolph et al., Chromatogr. Sci., 51 (HPLCBiol. Macromol.), 333-50 (1990).

N-terminal amino acid sequences of each subunit were determined usingmethods well known in the art. See, for example, Matsudaira, P., MethodsEnzymol., 182 (Guide Protein Purif.), 602-13 (1990). Fragments of eachsubunit were generated and partial amino acid sequences of the fragmentswere determined. Partial sequences of the α and β subunits of thisnitrile hydratase are shown in SEQ ID NOs.:5-9 and 10-13, respectively.

B. DNA Probe fQr Isolation of the Nitrile Hydratase Gene:

In order to isolate the nitrile hydratase gene, a series of degenerate21-mer oligonucleotide primers based on the available NRRL-18668 NHaseamino acid sequence were designed and synthesized for use as polymerasechain reaction (PCR) primers. Genomic DNA was isolated from P. putida(NRRL-18668) by standard methods (Sambrook, J., et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989)) and was used as a target for PCR with numerousdegenerate primer combinations. The resulting amplified products weresubjected to Southern analysis (Southern, E. M., J. Mol. Biol., 98, 503,(1975)) using isolated Rhodococcus rhodochrous J1 L-NHase gene(Kobayashi, M., Biochem. Biophys. Acta 1129:23-33 (1991)) as a probe.One strongly hybridizing fragment of 0.7 kb was identified from a PCRreaction based on the degenerate primers designated D1 and D7. Thesequences of D1 and D7 are identified in the Sequence Listing as SEQ IDNO.:14 and SEQ ID NO.:15, respectively. The 0.7 kb PCR fragment wassubcloned into the plasmid M13 using standard methods (Sambrook, supra)and sequenced. Sequencing revealed that the 0.7 kb fragment demonstrateda 60% base homology to the Rhodococcus rhodochrous J1 L-NHase gene.Deduced amino acid sequence from this 0.7 kb fragment was compared toavailable NRRL-18668 amino acid sequences determined previously and toother known NHase sequences. The comparison confirmed that this fragmentwas part of the P. putida NHase gene. The 0.7 kb DNA fragment wassequenced and is identified as SEQ ID No.:16. The 0.7 kb fragment wasused as a probe to isolate a genomic DNA fragment from NRRL-18668 whichcontains the entire NHase gene.

C. Isolation of a Genomic DNA Fragment Containing NRRL-18668 NHase Gene:

Genomic DNA isolated from P. putida (NRRL-18668) was digested withrestriction enzymes EcoR1 and Xho1 and size-selected by agarose gelelectrophoresis based on Southern blotting using the 0.7 kb DNA fragmentdescribed above as a probe. Restricted genomic DNA was then cloned intophage lambda ZAPII Stratagene, La Jolla, Calif.!. The lambda library wasscreened with the 0.7 kb DNA fragment probe and one positivelyhybridizing phage clone with a DNA insert of 6.5 kb was identified andisolated.

D. Plasmid Construction and Host Transformation and Confirmation ofNHase Sequence:

Once a positive clone containing a 6.5 kb insert was identified, thepresence of the NHase gene in the clone was confirmed by a process of(i) constructing a plasmid containing the 6.5 kb insert (pSW1, FIG. 1);(ii) transforming a suitable host cell with this plasmid; (iii) growingup the transformed host and purifying the plasmid DNA; (iv) constructinga restriction map from the purified DNA (FIG. 2); and (v) sequencing theNHase genes. The confirmation process is common and well known in theart and techniques used may be found in Sambrook supra.

Sequence analysis confirmed the nitrile hydratase coding regions, whichconsisted of two open reading frames corresponding to the alpha and betasubunits of the corresponding NHase protein as defined in the SequenceListing by SEQ ID NO.:17 and FIG. 7. The α and β open reading frameswere analyzed for base sequence similarly to the Rhodococcus rhodochrousJ1 L-NHASE gene used as a probe and described above. Homologycomparisons showed that the α open reading frame had 64% homology to theregion encoding the α subunit on the J1 gene and the β open readingframe had 52% homology to the region encoding the β subunit on the J1gene.

II. CONSTRUCTION OF EXPRESSION VECTOR AND EXPRESSION STRAINS

The present invention provides a transformed host cell capable ofexpressing active nitrile hydratase enzyme. Generally, it is preferredif the host cell is an E. coli, however, it is not outside the scope ofthe invention to provide alternative hosts. Such alternative hosts mayinclude, but are not limited to, members of the genera Pseudomonas,Rhodococcus, Acinetobacter, Bacillus, Saccharomyces, Pichia,Aspergillus, Hansenula, and Streptomyces.

The present invention provides a variety of plasmids or vectors suitablefor the cloning of the nitrile hydratase gene in the desired host.Suitable vectors for construction contain a selectable marker andsequences allowing autonomous replication or chromosomal integration.Additionally, suitable vectors for expression contain sequencesdirecting transcription and translation of the heterologous DNAfragment. These vectors comprise a region 5' of the heterologous DNAfragment which harbors transcriptional initiation controls, andoptionally a region 3' of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the host cell, althoughsuch control regions need not be derived from the genes native to thespecific species chosen as a production host. Suitable vectors can bederived, for example, from a bacteria (e.g., pET, pBR322, pUCl9, pSP64,pUR278 and pORFl), a virus (such as bacteriophage T7 or a M-13 derivedphage), a cosmid, a yeast or a plant. Protocols for obtaining and usingsuch vectors are known to those in the art. (Sambrook, supra.)

Vectors suitable for E. coli will have compatible regulatory sequencesand origins of replication. They will be preferably multicopy and have aselectable marker gene, for example, a gene coding for antibioticresistance.

Promoters useful for driving the expression of heterologous DNAfragments in E. coli are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving the gene encoding thenitrile hydratase enzyme is suitable for the present invention, althoughpromoters native to E. coli are preferred and the inducible IPTG Ptacpromoter is most preferred (deBoer, H., Proc. Natl. Acad. Sci. USA,80:21-25 (1983). Although an inducible promoter is preferred, one ofskill in the art will appreciate that either inducible or constitutivepromoters are suitable.

Within the context of the present invention the entire 6.5 kb DNA insertcontaining the NRRL-18668 NHase gene in the plasmid pSW1 was subclonedinto the wide-host-range vector pMMB207 (Bagdasarian, M., Gene, 97:39-47(1991)) under the control of the Ptac promoter to create an expressionvector designated pSW2 (FIG. 3). Additionally, the 2.8 kb Pst1 DNAfragment derived from the 6.5 kb DNA fragment and containing theNRRL-18668 NHase gene but with substantially less upstream anddownstream flanking sequence, was also subcloned into the vector pMMB207under the control of the Ptac promoter to generate the plasmid pSW5(FIG. 4). Comparing these two expression constructs allowed Applicantsto investigate proximal accessory sequences or proteins which might beinvolved in expression or activity of NHase. Applicants' studiesindicated that the NHase genes may be part of an operon which generatesa 10 kb mRNA transcript, of which only approximately 1.5 kb is accountedfor by NHase. This suggests that additional genes are encoded by theupstream and downstream sequence flanking NHase. Others have described arequirement for downstream sequence for efficient expression of NHase inRhodococcus sp. N-774 (Hashimoto, Y., Biosci. Biotech. Biochem.,58:1859-1865 (1994)).

Following cloning, E. coli XL1-Blue host was transformed in parallelwith the plasmid pSW2 or pSW5 described above. Methods of transforminghost cells with foreign DNA are common and well known in the art. Forexample, transforming host cells with foreign DNA may be accomplishedusing calcium-permeabilized cells, electroporation, or by transfectionusing a recombinant phage virus. (Sambrook supra). Plasmid DNA wasisolated from these transformants and enzyme restriction analysisconfirmed the construction of two separate strains, one harboring thepSW2 plasmid and the other harboring the pSW5 plasmid.

The gene encoding the α subunit, and the gene encoding the β subunit ofNRRL-18668 NHase were also expressed in an alternative host, themethylotrophic yeast Pichia pastoris. Methods for producing heterologousproteins in P. pastoris are well known in the art. For each subunit, thecoding sequence was placed under control of the methanol induciblepromoter, alcohol oxidase I (AOX1), in a vector which was subsequentlyintegrated into the host chromosome. Each subunit was produced in therespective host after induction by methanol. NHase activity was notreproducibly obtained upon mixing extract prepared from the α producingstrain with extract prepared from the β producing strain. In addition, asingle strain producing both α and β subunits under control of the AOX1promoter was constructed. Both subunits were produced in thisrecombinant P. pastoris strain, but NHase activity was not obtained.

Applicants sequenced DNA both upstream and downstream of the NHasegenes, and identified at least two open reading frames, one upstream andone downstream. The upstream open reading frame was determined to encodean amidase enzyme, based on comparison of the deduced amino acidsequence to other amidase amino acid sequences. Plasmids wereconstructed for the expression of NRRL-18668 amidase in E. coli. Asearch of the protein database with the deduced amino acid sequenceencoded by the downstream open reading frame (designated P14K) indicatedno significant matches. Plasmids were constructed for expression ofNHase genes only or NHase and P14K genes in both E. coli and P.pastoris. In both E. coli and P. pastoris, NHase activity was obtainedonly when P14K was co-expressed with the NHase genes. The preference forhydrolysis of S-nitriles (stereo-specificity) observed in the nativeorganism was also demonstrated in the recombinant orgamisms producingactive NHase.

III. EXPRESSION OF THE NITRILE HYDRATASE ENZYME AND CONVERSION OFSUBSTRATES

Transformed E. coli cells harboring plasmid pSW2 under the control ofthe IPTG inducible Ptac promoter, were grown under standard conditionsand induced to express the nitrile hydratase enzyme. Cells wereharvested and lysed and the protein was detected in crude lysates bySDS-polyacrylamide gel electrophoresis followed by western blot analysis(Egger et al., Mol. Biotechnol., 1(3), 289-305 (1994)) using antiseraraised against NRRL-18668 NHase protein (FIG. 5). Under these conditionsinduced cells produced approximately 10-fold as much nitrile hydrataseprotein as uninduced cells. Nitrile hydratase was not detected from acontrol strain harboring the vector pMMB207 without the 6.5 kb insert.

Nitrile hydratase is typically confirmed by incubating a suitablesubstrate nitrile in the presence of the crude or purified enzyme.Suitable substrates for the instant hydratase include a variety ofracemic alkyl nitrites such as methacrylonitrile, methylbutyronitrileand propionitrile. In the instant case, nitrile hydratase activity wasconfirmed by monitoring the conversion of methacrylonitrile to thecorresponding amide. Induced cells harboring the plasmid pSW2 showedrapid conversion of methacrylo-nitrile, while induced cells without thepSW2 plasmid showed no conversion of methacrylonitrile. Additionally,induced cells harboring the plasmid pSW5 show no conversion ofmethacrylonitrile.

Stereospecific activity of the enzyme produced in induced cellsharboring plasmid pSW2 was confirmed by monitoring the conversion ofR,S-CPIN to amide products using reverse-phase or chiral high pressureliquid chromatography (HPLC). Methods of enantiomer separation on HPLCare well known in the art. See, for example, Mutton, I., Pract. ApproachChiral Sep., Liq. Chromatogr., 329-55 (1994), Editor(s): Subramanian,Ganapathy, Publisher: VCH, Weinheim, Germany.

IV. CO-EXPRESSION OF NITRILE HYDRATASE AND AMIDASE

The present invention further provides a transformed microorganismcapable of co-expressing both a heterologous nitrile hydratase gene anda heterologous amidase gene. This transformant is capable of effectingthe conversion of racemic mixtures of aryl-2-alkane nitriles to thecorresponding carboxylic acids via the amide intermediate.

A number of amidase encoding genes may be suitable for co-expressionwith the instant nitrile hydratase. However, the amidase gene isolatedfrom Pseudomonas chlororaphis B23 and defined above is preferred.

The gene encoding the Pseudomonas chlororaphis B23 amidase is known(Nishiyama, M. J., Bacteriol., 173:2465-2472 (1991)) and was obtainedthrough PCR amplification using appropriate primers. The amplified genecomprising 1.5 kb was subcloned into a pMMB207 plasmid (alreadycontaining the nitrile hydratase gene) using standard restriction enzymedigestion and ligation techniques (Sambrook supra) to generate theplasmid pSW17 (FIG. 6). The plasmid pSW17 was constructed so as to placethe amidase gene and the nitrile hydratase gene both under the controlof the same IPTG inducible Ptac promoter. The plasmid pSW17 was thenused to transform a suitable host cell (e.g., E. coli XL1-Blue)according to standard methods.

In order to confirm the activity of the amidase produced in cellstransformed with plasmid pSW17, cells transformed by plasmid pSW17 weregrown up and induced with IPTG in the presence of a suitable nitrile andthe chiral amide and free acid products were identified by chiral HPLCanalysis.

The following Examples are meant to illustrate the invention but shouldnot be construed as limiting it in any way.

EXAMPLE 1 Isolation, Purification, and Amino Acid Sequencing of Portionsof the Nitrile Hydratase α and β Subunits

Pseudomonas putida (NRRL-18668) was cultured in a medium (10 g/Lglucose, 8.7 g/L K₂ HPO₄, 6.8 g/L KH₂ PO₄, 2.0 g/L acetonitrile, 1.85g/L NaNO₃, 0.50 g/L MgSO₄.7H₂ O, 0.050 g/L FeSO₄.7H₂ O, 0.30 mg/LMnCl₂.4H₂ O, 0.10 mg/L H₃ BO₃, 0.050 mg/L NiSO₄.6H₂ O, 0.050 mg/LCuSO₄.5H₂ O, 0.050 mg/L Co(NO₃)₂.6H₂ O, 0.030 mg/L Na₂ MoO₄.2H₂ O, 0.030mg/L ZnSO₄.4H₂ O, 0.020 mg/L KI, 0.020 mg/L KBr, 0.010 mg/Lpyridoxine.HCl, 0.0050 mg/L thiamine.HCl, 0.0050 mg/L D-pantothenate,Ca²⁺ salt, 0.0050 mg/L riboflavin, 0.0050 mg/L nicotinic acid, 0.0050mg/L p-aminobenzoic acid, 0.0020 mg/L biotin, 0.0020 mg/L vitamin B₁₂,0.0020 mg/L folic acid, pH 7.0) at 30° C. for 48 h. The bacterial cellswere harvested. 100 g of the bacterial cells were disrupted and the cellfree extract fractionated with ammonium sulfate. The ammonium sulfatefractionation precipitate was dissolved in buffer and loaded on a PhenylSepharose CL-4B chromatography column (Pharmacia Biotech, Uppsala,Sweden), followed by a DEAE-cellulose chromatography column, and asecond DEAE-cellulose chromatography column (Whatman, Maidstone,England). Active fractions were pooled and concentrated. The concentratecontaining the enzyme was loaded on a reverse phase high performancechromatography column (Vydac 208TP104) and two subunits (α and β) wereobtained. The N-terminal amino acid sequence of the α- and β-subunitswas determined using an amino acid sequencer (Beckman model LF3000G gasphase protein sequencer, Fullerton, Calif. The α- and β-subunits werecleaved separately using cyanogen bromide, TPCK-treated trypsin, andAspN protease, and the peptides generated were separated on a reversephase high performance chromatography column (Vydac 208TP104, TheSeparations Group, Hesperia, Calif.). Fractions containing well-resolvedpeptides were sequenced using the same technique. The sequences of theindividual peptides were combined into partial sequences of the subunitsby alignment with the published sequences of the α- and β-subunits ofnitrile hydratases from P. chlororaphis B23 Nishiyama et al., J.Bacteriol., 173:2465-2472 (1991)!, Rhodococcus N-774 Ikehata et al.,Eur. J. Biochem., 181:563-570 (1989)!, and Rhodococcus rhodochrous J1Kobayashi et al., Biochim. Biophys. Acta, 1129:23-33 (1991)!. Thepartial sequences of the of the α- and β-subunits of nitrile hydratasefrom Pseudomonas putida (NRRL-18668) were identified as defined in theSequences Listing as SEQ ID NOs.:5-9 and SEQ ID NOs.:10-13,respectively.

EXAMPLE 2 Preparation of DNA Probe for NRRL-18668 NHase Gene

The degenerate oligonucleotide designated Dl as defined in the SequenceListing as SEQ ID NO.:14, and the degenerate oligonucleotide designatedD7 as defined in the Sequence Listing as SEQ ID NO.:15 were used asprimers in a polymerase chain reaction (PCR) Mullis, K. B., Meth.Enzymol., 155:335-350 (1987)! with NRRL-18668 genomic DNA as target. PCRconditions were as follows: 100 ng target, 1 μM each primer, 200 μM eachof dATP, dCTP, dGTP, dTTP, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mMMgCl₂, 0.001% gelatin, 25 U/mL Amplitaq™ DNA polymerase (Perkin ElmerCetus, Norwalk, Conn.). PCR parameters were as follows: 94° C. 1 min,55° C. 1 min, 72° C. 1 min, 40 cycles. One half of the PCR product wassubjected to ethidium bromide agarose gel electrophoresis followed bytransfer to nitrocellulose and Southern analysis with ³² P labeledRhodococcus rhodochrous J1 L-NHase gene as probe Southern, E. M., J.Mol. Biol., 98:503 (1975)!. Strong hybridization of a DNA fragment ofapproximately 0.7 kb suggested the presence of at least a portion of aNHase gene in this PCR product. The remaining half of the PCR productwas restricted with EcoR1 (the primers were designed with EcoR1 sites atthe 5' ends) and ligated to EcoR1 restricted M13 mp19 vector DNA.Ligation mix was used to transfect competent E. coli XL1-Blue which wasplated onto LB plates supplemented with IPTG and X-gal(5-bromo-4chloro-3indolyl-β-D-galactopyranoside) Maniatis, T., MolecularCloning: A Laboratory Manual (1989)!. Phage DNA was prepared fromseveral "white" plaques Maniatis, T., Molecular Cloning: A LaboratoryManual (1989)! and sequenced by dideoxy termination protocol usinguniversal primer Sanger, F., Science, 214:1205-1210 (1981)!. Analysis ofthe nucleotide sequence obtained as defined in the Sequence Listing asSEQ ID NO.:16 confirmed that the PCR product corresponds to part of theNHase gene.

EXAMPLE 3 Isolation of Genomic DNA Fragment Containing NRRL-18668 NHaseGene

Total genomic DNA (10 pg) from NRRL-18668 was isolated Maniatis, T.,Molecular Cloning: A Laboratory Manual (1989)!, restricted with EcoR1and Xho1, and one half subjected to agarose gel electrophoresis followedby Southern blot using the ³² P labeled 0.7 kb fragment described inExample 2 as a probe Southern, E. M., J. Mol. Biol., 98:503 (1975)!. Astrongly hybridizing band of approximately 6.5 kb was identified,suggesting that the NHase gene (or part of it) resides on this 6.5 kbgenomic DNA fragment. A duplicate agarose gel was run and a gel slicefrom the 6.5 kb region was excised. DNA extracted from the gel sliceisolated Maniatis, T., Molecular Cloning: A Laboratory Manual (1989)!was ligated to lambda DNA restricted with EcoR1 and Xho1. The ligationmix was packaged into phage particles and used to transfect E. coliXL1-Blue according to the manufacturer's instructions Stratagene, LaJolla, Calif.!. Several thousand plaques were screened using the ³²P-labeled 0.7 kb fragment as probe Maniatis, T., Molecular Cloning: ALaboratory Manual (1989)!. One positively hybridizing plaque wassubsequently purified.

EXAMPLE 4 Construction of Plasmid Containing NRRL-18668 NHase Gene

DNA from the purified phage plaque described in Example 3 was excisedand converted to a pBluescript-based plasmid according the themanufacturer's instructions Stratagene, La Jolla, Calif.!, anddesignated pSW1. The plasmid pSW1 has a 6.5 kb insert containing theNRRL-18668 NHase gene as described in FIG. 1.

EXAMPLE 5 Transformation of Host by Plasmid Containing NRRL-18668 NHaseGene

The plasmid pSW1 described in Example 4 was used to transform competentE. coli XL1-Blue cells by the CaCl₂ method Maniatis, T., MolecularCloning: A Laboratory Manual (1989)!.

EXAMPLE 6 Recombinant Plasmid Purification and Construction ofRestriction Map for Genomic DNA Fragment Containing NRRL-18668 NHaseGene

Plasmid DNA purified by the alkaline lysis method Maniatis, T.,Molecular Cloning: A Laboratory Manual (1989)! from E. coli cellsharboring plasmid pSW1, described in Example 5, was restricted withEcoR1, Pst1, Kpn1, Hind3, and Xho1 singly or in various combinations,followed by agarose gel analysis, and Southern analysis using the 0.7 kbPCR product described in Example 2 as a probe Southern, E. M., J. Mol.Biol., 98:503 (1975)!. A restriction map constructed for the 6.5 kbinsert fragment of the plasmid pSW1, including the location of the NHasegene is shown in FIG. 2.

EXAMPLE 7 DNA Sequencing of NRRL-18668 NHase Gene

Based on the restriction map described in Example 6, the nucleotidesequence of a fragment of DNA encompassing the NHase gene was determinedby the Sanger dideoxy method Sanger, F., Science, 214:1205-1210 (1981)!using double-stranded plasmid DNA as template. The nucleotide sequenceand the corresponding predicted amino acid sequences for the α and βpeptides are defined in the Sequence Listing as SEQ ID NO.:17 and FIG.7.

EXAMPLE 8 Construction of NRRL-18668 NHase Expression Vector

Plasmid pSW1 was restricted with EcoR1 and Xho1 and the 6.5 kb fragmentwas ligated to the wide host range plasmid pMMB207 Bagdasarian, M.,Gene, 97:39-47 (1991)! restricted with EcoR1 and Sal 1 to generate theplasmid designated pSW2 and shown in FIG. 3. The 2.8 kb Pst1 DNAfragment containing the NRRL-18668 NHase gene was excised from plasmidpSW2 by digestion with Pstl restriction enzyme and ligated into the Pst1site of vector pMMB207 to generate the plasmid designated pSW5 and shownin FIG. 4.

EXAMPLE 9 Construction of NRRL-18668 NHase Expression Strain

Plasmids pSW2 and pSW5 described in Example 8 were used to transformcompetent E. coli XL1-Blue cells which were plated onto LB platessupplemented with 12.5 μg/mL chloramphenicol Maniatis, T., MolecularCloning: A Laboratory Manual (1989)!.

EXAMPLE 10 Expression of NRRL-18668 NHase Protein

E. coli cells harboring plasmid pSW2, described in Example 8A, weregrown in SOC media (0.5 g/L NaCl, 20 g/L bacto-tryptone, 5 g/Lbacto-yeast extract, 20 mM glucose, 2.5 mM KCl, 10 mM MgCl₂) at 37° C.to OD600=0.5, followed by induction at 30° C. by the addition of IPTG to1 mM. After induction times ranging from 0.5 h to 3 h, cells wereharvested by centrifugation, and suspended in 1/10 volume PBS (8.0 g/LNaCl, 0.2 g/L KCl, 1.44 g/L Na₂ HPO₄, 0.24 g/L KH₂ PO₄ pH 7.4). A cellsuspension equivalent to 0.05 OD600 units is added to an equal volume of2× SDS gel-loading buffer (100 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.2%bromophenol blue, 20% glycerol), boiled for 5 min, and analyzed by SDSPAGE Laemmli, U.K., Nature, 227:680-685 (1970)! followed by western blotTowbin, H., Proc. Natl. Acad. Sci., 76:4350-4354 (1979)! using antiseraraised against NRRL-18668 NHase protein. A positive signal was obtainedat approximately 28 kd and corresponded to purified NHase protein asshown in FIG. 5.

EXAMPLE 11 Expression of Active NRRL-18668 NHase

E. coli cells harboring plasmid pSW2, described in Eample 9, were grownand induced as described in Example 9 in a 500 mL batch. Cells wereharvested by centrifugation and washed with pH 7.2, 0.1M phosphatebuffer(KH₂ PO₄ adjusted with 50% NaOH) containing 15% glycerol. Washedcells were stored frozen at -70° C. Washed and frozen E. coli cellsharboring the pSW2 plasmid and were suspended in 100 mM phosphatebuffer, pH 7, at a cell density of O.D.₄₉₀ =0.62. Methacrylo-nitrile wasadded to a final concentration of 10 mM and the mixture was shaken at250 rpm at room temperature. Analysis of supernatant showed thatmethacrylonitrile was rapidly converted to hydrolysis products after 30min. Cells without the pSW2 plasmid showed no activity.

EXAMPLE 12 Production of Chiral Amides

Induced E. coli cells harboring the pSW2 plasmid and producingstereospecific nitrile hydratase activity as described in Example 11were suspended in 100 mM phosphate buffer, pH 7, and a concentration of50 mg/mL. One milliliter of this suspension was placed in a glass vialcontaining 19.3 mg of R,S-CPIN. The suspension was shaken at 250 rpm ona rotary shaker at room temperature for 68 h. Analysis by chiral HPLCreveals only the S-CPIAm was produced from the R,S-CPIN.

    ______________________________________           mg nitrile   mg amide    Time, h  R-CPIN  S-CPIN     R-CPIAm                                       S-CPIAm    ______________________________________    0        9.6     9.6        0      0    68       9.6     5.5        0      4.5    ______________________________________

EXAMPLE 13 Construction of a Vector for Co-Expression Of NRRL-18668NHase and Pseudomonas Chlororaphis B23 Amidase

The amidase gene from Pseudomonas chlororaphis B23 (defined as SEQ IDNO.:20) was obtained through PCR amplification using primers withoverhanging 5' EcoR1 sites as defined in the Sequence Listing as SEQ IDNO.:18 and SEQ ID NO.:19. This 1.4 kb DNA fragment containing the B23amidase gene was digested with EcoR1 restriction enzyme and ligated intothe EcoR1 site of pMMB207, and the 5.0 kb EcoR1/Hind111 DNA fragmentfrom pSW1, described in Example 4, was subcloned between the Xba1 andHind111 to generate the plasmid pSW17 as shown in FIG. 6.

EXAMPLE 14 Construction of Strain for Co-Expression of NRRL-18668 NHaseand Pseudomonas Chlororaphis B23 Amidase

Plasmid pSW17 described in Example 13 was used to transform competent E.coli XL1-Blue cells which were selectively grown on LB platessupplemented with 12.5 μg/mL chloramphenicol Maniatis, T., MolecularCloning: A Laboratory Manual (1989)!.

EXAMPLE 15 Comparison of NHase Activity from pSW2 and pSW5

E. coli cells harboring the pSW2 or pSW5 plasmid and induced accordingto the protocol in Example 11 were each suspended separately in 100 mMphosphate buffer, pH 7, at a concentration of 20 mg/mL. Butyronitrilewas added to each suspension to a final concentration of 10 mM. Thesuspensions were shaken at 250 rpm on a rotary shaker at roomtemperature for 24 h. At the end of the incubation period, 0.1%phosphoric acid was added to the suspensions, bringing them to a pH of2-3 and stopping nitrile hydratase activity. Cells were removed from thesuspension by centrifugation. Analysis of the reactions showed thefollowing products:

pSW2--94% butyramide, 6% butyronitrile;

pSW5--<1% butyramide, 100% butyronitrile.

EXAMPLE 16 Production of S-CPIAm and S-CPIA from R,S-CPIN

E. coli cells harboring the pSW17 and induced according to the protocolin Example 11 were suspended in 100 mM phosphate buffer, pH 7, at aconcentration of 100 mg/mL. One milliliter of this suspension was placedin a glass vial containing 19.3 mg of R,S-CPIN dispersed in a dry formon 0.5 g of 0.5 mm glass beads. The suspension was shaken in a 20 mLscintillation vial at 250 rpm on a rotary shaker at room temperature for68 h. Analysis by chiral HPLC reveals both S-CPIAm and the S-CPIA wereproduced from the R,S-CPIN.

    ______________________________________    mg nitrile     mg amide      mg acid    Time, h           R-CPIN  S-CPIN  R-CPIAm                                  S-CPIAm                                         R-CPIA                                               S-CPIA    ______________________________________    0      9.6     9.6     0      0      0     0    68     9.6     8.4     0      0.84   0     0.42    ______________________________________

EXAMPLE 17 Nucleotide sequencing of DNA regions flanking NRRL-18668NHase gene

The nucleotide sequences of DNA regions flanking the NRRL-18668 NHasewere determined by the Sanger dideoxy method (Sanger, F. (1981) Science214:1205-1210) using double-stranded plasmid DNA as template. Using pSW1(FIG. 1) as template, the nucleotide sequence downstream of NHase, downto the Xho1 site (FIG. 2), was determined. This sequence contains atleast one gene, and potentially several more, which is defined as P14K,the nucleotide sequence of which is defined in Sequence Listing SEQ IDNO.:21, and the deduced amino acid sequence is defined in SequenceListing SEQ ID NO.:22. P14K is required for NHase activity as describedbelow.

The nucleotide sequence upstream of NHase, up to the EcoR1 (FIG. 2), wasdetermined using pSW1 (FIG. 1) as template. The nucleotide sequencefurther upstream of the EcoR1 site was determined after subcloning DNAfragments corresponding to this region as follows. NRRL-18668 genomicDNA was digested with Pst1 and then self-ligated. Oligo-nucleotideprimers designed to bind 3' to EcoR1 heading upstream (FIG. 2) and 5' toPstl heading downstream (FIG. 2), and defined as Sequence Listing SEQ IDNO.:23 and Sequence Listing SEQ ID NO.:24, respectively, were used in aPCR reaction to amplify a 0.8 kb fragment corresponding to DNA upstreamof the EcoR1 site (FIG. 8). NRRL-18668 genomic DNA was digested withEcoR1 and then self-ligated. Oligo-nucleotide primers designed to bind3' to Pst1 heading upstream (FIG. 8) and 5' to EcoR1 heading downstream(FIG. 8), and defined as Sequence Listing SEQ ID NO.:25 and SEQ IDNO.:26, respectively, were used in a PCR reaction to amplify a 0.7 kbfragment corresponding to DNA upstream of the Pst1 site (FIG. 9). Bysubcloning and sequencing the PCR fragments, the nucleotide sequenceupstream of NHase, up to the EcoR1 site (FIG. 9) was determined. Thissequence contains at least one gene, and potentially more, which hasbeen identified as encoding an amidase (based on homology to otheramidase sequences), the nucleotide sequence of which is defined asSequence Listing SEQ ID NO.:27, and the deduced amino acid sequencedefined as Sequence Listing SEQ ID NO.:28.

A compiled map of the entire 8.0 kb DNA fragment, indicating genesidentified, is shown in FIG. 10.

EXAMPLE 18 Construction of plasmids for expression of NRRL-18668 NHasein Pichia pastoris

The 0.9 kb EcoRl/Xbal fragment in pHIL-D4 (Phillips Petroleum,Bartlesville, Okla.) was replaced by the 0.9 kb EcoR1/Xba1 fragment frompAO815 (Invitrogen, San Diego, Calif.) to generate the plasmid pHIL-D4B2(FIG. 11) which contains the following elements: 5'AOX1, P. pastorismethanol inducible alcohol oxidase I (AOX1) promoter; AOX1 term, P.pastoris AOX I transcriptional termination region; HIS4, P. pastorishistidinol dehydrogenase-encoding gene for selection in his4 hosts; kan,sequence derived from transposon Tn903 encoding aminoglycoside3'-phosphotransferase, conferring kanamycin, neomycin and G418resistance in a wide variety of hosts, and useful as an indicator ofcassette copy number; 3'AOX1, P. pastoris sequence downstream from AOX1,used in conjunction with 5'AOX1 for site-directed vector integration;ori, pBR322 origin of DNA replication allowing plasmid manipulations inE. coli; and amp, β-lactamase gene from pBR322 conferring resistance toampicillin. An additional feature of PHIL-D4B2 is that multipleexpression cassettes (5'AOX1-gene-AOX1term) can easily be placed intoone plasmid by subcloning cassettes on Bg12/Xba1 fragments intoBamH1/Xba1 sites.

The genes encoding α, β, and P14K (FIG. 10) were PCR amplified usingprimers with EcoR1 sites at the 5' ends. The PCR products were digestedwith EcoR1, and subcloned into the EcoR1 site of pHIL-D4B2 to generatepSW46 (FIG. 12), pSW47 (FIG. 13) and pSW48 (FIG. 14), respectively. TheBg12/Xba1 fragment from pSW47 containing the β expression cassette wassubcloned into the BamH1/Xba1 sites of pSW46 to generate pSW49 (FIG.15), which contains expression cassettes for α and β. The Bg12/Xba1fragment from pSW48 containing the P14K expression cassette wassubcloned into the BamH1/Xba1 sites of pSW49 to generate pSW50 (FIG.16), which contains expression cassettes for α, β and P14K.

EXAMPLE 19 Construction of Pichia pastoris strain for expression ofNRRL-18668 NHase

P. pastoris strain GTS115(his4) (Phillips Petroleum, Bartlesville,Okla.) was transformed with 1-2 μg of Bg12-linearized plasmid pSW49 or1-2 μg of Bgl2-linearized plasmid pSW50 using the spheroplasttransformation method as described (Cregg et al. (1985) Mol. Cell. Biol.5: 3376-3385). Cells were regenerated on plates without histidine for3-4 d at 30° C. All transformants arise after integration of plasmid DNAinto the chromosome. Chromosomal DNA was prepared from his⁺transformants and subjected to PCR analysis with primers specific for α,β and P14K genes. An isolated pSW49 transformant positive for α and βgenes, and an isolated pSW50 transformant positive for α, β and P14Kgenes, designated SW49 and SW50.2, respectively, were selected forfurther study. P. pastoris strain SW50.2 was deposited with ATCC andassigned accession number ATCC 74391.

EXAMPLE 20 NRRL-18668 NHase activity in engineered P. pastoris

P. pastoris strains SW49 and SW50.2 were grown to A₆₀₀ of 2-10 in MGY(1.34% yeast nitrogen base without amino acids, 0.00004% biotin, 1%glycerol) with shaking at 30° C. Cells are then pelleted and induced byresuspending in MM (1.34% yeast nitrogen base without amino acids,0.00004% biotin, 0.5% methanol) and incubated with shaking at 30° C. for1-4 d. Cells were harvested by centrifugation and washed in PBS (0.1MKH₂ PO₄, pH 7.2). NHase activity was demonstrated by methacrylonitrileassay, in which cells were resuspended in PBS at A₆₀₀ of 0.6, andmethacrylonitrile was added to a final concentration of 10 mM. Afterincubation with shaking at room temperature, conversion ofmethacrylonitrile to methacrylamide by NHase was demonstrated bymonitoring the increase in A₂₂₄ of the supernatant. Cells boiled beforeassay serve as a negative control. NHase activity was observed in SW50.2which harbors expression cassettes for α, β and P14K, while SW49, whichonly harbors expression cassettes for α and β showed negligible NHaseactivity.

    ______________________________________    A.sub.224    rxn time, min               SW49        SW50.2  SW50.2 boil    ______________________________________    0          0.260       0.360   0.110    15         0.360       1.390   0.125    ______________________________________

Stereospecific NHase activity was also demonstrated in induced SW50.2cells by using R-2-(4-chlorophenyl)-3-methylbutyronitrile (R-CPIN) orS-2-(4-chlorophenyl)-3-methylbutyronitrile (S-CPIN) as substrate and andthen analyzing for conversion to the corresponding amides (R-CPIAm andS-CPIAm, respectively) by HPLC.

    ______________________________________    mM    rxn time, h               R-CPIN  R-CPIAm    S-CPIN                                        S-CPIAm    ______________________________________    0          10      0          10    0    48         10      0          5.5   4.5    ______________________________________

Bioconversion of adiponitrile (ADN) to 5-cyanovaleramide (5-CVAm) wasalso demonstrated in permeabilized SW50.2 cells, and in SW50.2 cellextracts. Permeabilized cells were prepared by the addition ofbenzalkonium chloride (Lonza Baequat MB-50) to a 10% (wt) suspension ofinduced cells to yeild 1% (wt MB-50:wt cells). The suspension was thenmixed on a nutator mixer for 60 min at room temperature, after whichcells were washed by centrifugation 3 times with 50 mM phospahte buffer,pH 7.0. Extracts were prepared by rapidly vortexing induced cells with0.5 mm glass beads (BioSpec Products) in 50 mM KH₂ PO₄, pH 7.0/1 mMEDTA/0.1 mM PMSF for 2 min. NHase activity was determined to be 34-38U/g wet wt (permeabilized cells), and 35-56 U/g wet wt (cell extracts).

EXAMPLE 21 Construction of plasmid for expression of NRRL-18668 amidasein E. coli

The gene encoding NRRL-18688 amidase was PCR amplified using an upstreamprimer with a Hind3 site at the 5' end and a downstream primer with anXho1 site at the 5' end. The PCR product was subcloned into the vectorpET-21a(+) (Novagen, Madison, Wis.) between the Hind3 and Xho1 sites togenerate the expression plasmid pSW37 (FIG. 17).

EXAMPLE 22 Construction of E. coli strain for expression of NRRL-18668amidase

E. coli strain BL21(DE3) (Novagen, Madison, Wis.) was transformed withpSW37 using the calcium chloride procedure (Maniatis et al. (1989)Molecular Cloning: A Laboratory Manual), and an isolated transformantwas designated SW37, and deposited with ATCC and assigned accessionnumber ATCC 98174. Induced SW37 shows production of amidase enzyme basedon Coomassie Blue stained denaturing polyacrylamide gel electrophoresisof soluble cell extract.

EXAMPLE 23 NRRL-18668 amidase activity in engineered E. coli

E. coli strain SW37 is grown in LB media at 30° C. to A₆₀₀ =0.5, atwhich time IPTG is added to 1 mM and incubation continued for 2 h. Cellsare then pelleted and washed in PBS. Cells are incubated with 10 mMbutyramide and conversion to butyric acid is monitored by HPLC.

EXAMPLE 24 Construction of plasmid for expression of NRRL-18668 amidaseand NHase in E. coli

The entire 8.0 kb DNA fragment (shown in FIG. 10) was subcloned betweenthe EcoR1 and Xho1 sites of the vector pET-21(+) (Novagen, Madison,Wis.) to generate the plasmid pSW23 (FIG. 18).

EXAMPLE 25 Construction of E. coli strain for co-expression ofNRRL-18668 amidase and NHase

E. coli strain BL21(DE3) (Novagen, Madison, Wis.) was transformed withpSW23 using the calcium chloride procedure (Maniatis et al. (1989)Molecular Cloning: A Laboratory Manual), and an isolated transformantwas designated SW23, and deposited with ATCC and assigned accessionnumber ATCC 98175. Induced SW23 shows production of NHase enzyme andamidase enzyme based on Coomassie Blue stained denaturing polyacrylamidegel electrophoresis of soluble cell extract.

EXAMPLE 26 NRRL-18668 amidase and NHase activity in engineered E. coli

E. coli strain SW23 is grown in LB media at 30° C. to A₆₀₀ =0.5, atwhich time IPTG is added to 1 mM and incubation continued for 2 h. Cellsare then pelleted and washed in PBS. Cells are incubated with 10 mMbutyronitrile and conversion to butyric acid is monitored by HPLC.Stereospecific conversion of S-CPIN, relative to R-CPIN, to thecorresponding acid (S-CPIAc) can also be monitored by HPLC.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 28    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 210 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    MetGlyGlnSerHisThrHisAspHisHisHisAspGlyTyrGlnAla    151015    ProProGluAspIleAlaLeuArgValLysAlaLeuGluSerLeuLeu    202530    IleGluLysGlyLeuValAspProAlaAlaMetAspLeuValValGln    354045    ThrTyrGluHisLysValGlyProArgAsnGlyAlaLysValValAla    505560    LysAlaTrpValAspProAlaTyrLysAlaArgLeuLeuAlaAspAla    65707580    ThrAlaAlaIleAlaGluLeuGlyPheSerGlyValGlnGlyGluAsp    859095    MetValIleLeuGluAsnThrProAlaValHisAsnValPheValCys    100105110    ThrLeuCysSerCysTyrProTrpProThrLeuGlyLeuProProAla    115120125    TrpTyrLysAlaAlaAlaTyrArgSerArgMetValSerAspProArg    130135140    GlyValLeuAlaGluPheGlyLeuValIleProAlaAsnLysGluIle    145150155160    ArgValTrpAspThrThrAlaGluLeuArgTyrMetValLeuProGlu    165170175    ArgProGlyThrGluAlaTyrSerGluGluGlnLeuAlaGluLeuVal    180185190    ThrArgAspSerMetIleGlyThrGlyLeuProThrGlnProThrPro    195200205    SerHis    210    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 217 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    MetAsnGlyIleHisAspThrGlyGlyAlaHisGlyTyrGlyProVal    151015    TyrArgGluProAsnGluProValPheArgTyrAspTrpGluLysThr    202530    ValMetSerLeuLeuProAlaLeuLeuAlaAsnAlaAsnPheAsnLeu    354045    AspGluPheArgHisSerIleGluArgMetGlyProAlaHisTyrLeu    505560    GluGlyThrTyrTyrGluHisTrpLeuHisValPheGluAsnLeuLeu    65707580    ValGluLysGlyValLeuThrAlaThrGluValAlaThrGlyLysAla    859095    AlaSerGlyLysThrAlaThrArgValLeuThrProAlaIleValAsp    100105110    AspSerSerAlaProGlyLeuLeuArgProGlyGlyGlyPheSerPhe    115120125    PheProValGlyAspLysValArgValLeuAsnLysAsnProValGly    130135140    HisThrArgMetProArgTyrThrArgAlaLysTrpGlyGlnTrpSer    145150155160    SerThrMetValCysPheValThrProAspThrAlaAlaHisGlyLys    165170175    GlyGluGlnProGlnHisValTyrThrValSerPheThrSerValGlu    180185190    LeuTrpGlyGlnAspAlaSerSerProLysAspThrIleArgValAsp    195200205    LeuTrpAspAspTyrLeuGluProAla    210215    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 633 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    ATGGGGCAATCACACACGCATGACCACCATCACGACGGGTACCAGGCACCGCCCGAAGAC60    ATCGCGCTGCGGGTCAAGGCCTTGGAGTCTCTGCTGATCGAGAAAGGTCTTGTCGACCCA120    GCGGCCATGGACTTGGTCGTCCAAACGTATGAACACAAGGTAGGCCCCCGAAACGGCGCC180    AAAGTCGTGGCCAAGGCCTGGGTGGACCCTGCCTACAAGGCCCGTCTGCTGGCAGACGCA240    ACTGCGGCAATTGCCGAGCTGGGCTTCTCCGGGGTACAGGGCGAGGACATGGTCATTCTG300    GAAAACACCCCCGCCGTCCACAACGTCTTCGTTTGCACCTTGTGCTCTTGCTACCCATGG360    CCGACGCTGGGCTTGCCCCCTGCCTGGTACAAGGCCGCCGCCTACCGGTCCCGCATGGTG420    AGCGACCCGCGTGGGGTTCTCGCGGAGTTCGGCCTGGTGATCCCCGCCAACAAGGAAATC480    CGCGTCTGGGACACCACGGCCGAATTGCGCTACATGGTGCTGCCGGAACGGCCCGGAACT540    GAAGCCTACAGCGAAGAACAACTGGCCGAACTCGTTACCCGCGATTCGATGATCGGCACC600    GGCCTGCCAACCCAACCCACCCCATCTCATTAA633    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 654 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    ATGAATGGCATTCACGATACTGGCGGAGCACATGGTTATGGGCCGGTTTACAGAGAACCG60    AACGAACCCGTCTTTCGCTACGACTGGGAAAAAACGGTCATGTCCCTGCTCCCGGCCCTG120    CTCGCCAACGCGAACTTCAACCTCGATGAATTTCGGCATTCGATCGAGCGAATGGGCCCG180    GCCCACTATCTGGAGGGAACCTACTACGAACACTGGCTTCATGTCTTTGAGAACCTGCTG240    GTCGAGAAGGGTGTGCTCACGGCCACGGAAGTCGCGACCGGCAAGGCTGCGTCTGGCAAG300    ACGGCGACGCGCGTGCTGACGCCGGCCATCGTGGACGACTCGTCAGCACCGGGGCTTCTG360    CGCCCGGGAGGAGGGTTCTCTTTTTTTCCTGTGGGGGACAAGGTTCGCGTCCTCAACAAG420    AACCCGGTGGGCCATACCCGCATGCCGCGCTACACGCGGGCAAAGTGGGGACAGTGGTCA480    TCGACCATGGTGTGTTTCGTGACGCCGGACACCGCGGCACACGGAAAGGGCGAGCAGCCC540    CAGCACGTTTACACCGTGAGTTTCACGTCGGTCGAACTGTGGGGGCAAGACGCTTCCTCG600    CCGAAGGACACGATTCGCGTCGACTTGTGGGATGACTACCTGGAGCCAGCGTGA654    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    GlyGlnSerHisThrHisAspHisHisHisAspGlyTyrGlnAlaPro    151015    ProGluAspIleAlaLeuArgValLysAlaLeuGluSerLeu    202530    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    AspLeuValValGlnThrTyrGluHisLysValGlyPro    1510    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    AsnGlyAlaLysValValAlaLysAlaTrpValAspProAlaTyrLys    151015    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    AspProArgGlyValLeuAlaGluPheGly    1510    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    GlyLeuProThrGlnProThrProSerHis    1510    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 83 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    MetAsnGlyIleHisAspThrGlyGlyAlaHisGlyTyrGlyProVal    151015    TyrArgGluProAsnGluProValPheArgTyrAspTrpGluLysThr    202530    ValMetSerLeuLeuProAlaLeuXaaAlaAsnGlyAsnPheAsnLeu    354045    AspGluPheArgHisSerIleGluArgMetGlyProAlaHisTyrLeu    505560    GluGlyThrTyrTyrGluHisTrpLeuHisValPheGluAsnLeuLeu    65707580    ValGluLys    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    GlyGluHisProGlnHisValTyr    15    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    SerPheThrSerValGluLeuTrpGlyGlnAspAlaSerSerProLys    151015    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    ValAspLeuTrpAspAspTyrLeuGluProAla    1510    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    GGAATTCGAYCAYCAYCAYGA21    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    GGAATTCTTYTCCCARTCRTA21    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 726 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    GAATTCGATCACCATCACGACGGGTACCAGGCACCGCCCGAAGACATCGCGCTGCGGGTC60    AAGGCCTTGGAGTCTCTGCTGATCGAGAAAGGTCTTGTCGACCCAGCGGCCATGGACTTG120    GTCGTCCAAACGTATGAACACAAGGTAGGCCCCCGAAACGGCGCCAAAGTCGTGGCCAAG180    GCCTGGGTGGACCCTGCCTACAAGGCCCGTCTGCTGGCAGACGCAACTGCGGCAATTGCC240    GAGCTGGGCTTCTCCGGGGTACAGGGCGAGGACATGGTCATTCTGGAAAACACCCCCGCC300    GTCCACAACGTCTTCGTTTGCACCTTGTGCTCTTGCTACCCATGGCCGACGCTGGGCTTG360    CCCCCTGCCTGGTACAAGGCCGCCGCCTACCGGTCCCGCATGGTGAGCGACCCGCGTGGG420    GTTCTCGCGGAGTTCGGCCTGGTGATCCCCGCCAACAAGGAAATCCGCGTCTGGGACACC480    ACGGCCGAATTGCGCTACATGGTGCTGCCGGAACGGCCCGGAACTGAAGCCTACAGCGAA540    GAACAACTGGCCGAACTCGTTACCCGCGATTCGATGATCGGCACCGGCCTGCCAACCCAA600    CCCACCCCATCTCATTAAGGAGTTCGTCATGAATGGCATTCACGATACTGGCGGAGCACA660    TGGTTATGGGCCGGTTTACAGAGAACCGAACGAACCCGTCTTTCGCTACGACTGGGAAAA720    GAATTC726    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1440 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    CGGGAGCGCAATCTGCAAGGTGGCATTGGCCTTCAGTGTCGATGCCGAGTTGAAGTCGCT60    GTACCCCTTTTTTCAACCACACCAGGAGAACCGCACCATGGGGCAATCACACACGCATGA120    CCACCATCACGACGGGTACCAGGCACCGCCCGAAGACATCGCGCTGCGGGTCAAGGCCTT180    GGAGTCTCTGCTGATCGAGAAAGGTCTTGTCGACCCAGCGGCCATGGACTTGGTCGTCCA240    AACGTATGAACACAAGGTAGGCCCCCGAAACGGCGCCAAAGTCGTGGCCAAGGCCTGGGT300    GGACCCTGCCTACAAGGCCCGTCTGCTGGCAGACGCAACTGCGGCAATTGCCGAGCTGGG360    CTTCTCCGGGGTACAGGGCGAGGACATGGTCATTCTGGAAAACACCCCCGCCGTCCACAA420    CGTCTTCGTTTGCACCTTGTGCTCTTGCTACCCATGGCCGACGCTGGGCTTGCCCCCTGC480    CTGGTACAAGGCCGCCGCCTACCGGTCCCGCATGGTGAGCGACCCGCGTGGGGTTCTCGC540    GGAGTTCGGCCTGGTGATCCCCGCCAACAAGGAAATCCGCGTCTGGGACACCACGGCCGA600    ATTGCGCTACATGGTGCTGCCGGAACGGCCCGGAACTGAAGCCTACAGCGAAGAACAACT660    GGCCGAACTCGTTACCCGCGATTCGATGATCGGCACCGGCCTGCCAACCCAACCCACCCC720    ATCTCATTAAGGAGTTCGTCATGAATGGCATTCACGATACTGGCGGAGCACATGGTTATG780    GGCCGGTTTACAGAGAACCGAACGAACCCGTCTTTCGCTACGACTGGGAAAAAACGGTCA840    TGTCCCTGCTCCCGGCCCTGCTCGCCAACGCGAACTTCAACCTCGATGAATTTCGGCATT900    CGATCGAGCGAATGGGCCCGGCCCACTATCTGGAGGGAACCTACTACGAACACTGGCTTC960    ATGTCTTTGAGAACCTGCTGGTCGAGAAGGGTGTGCTCACGGCCACGGAAGTCGCGACCG1020    GCAAGGCTGCGTCTGGCAAGACGGCGACGCGCGTGCTGACGCCGGCCATCGTGGACGACT1080    CGTCAGCACCGGGGCTTCTGCGCCCGGGAGGAGGGTTCTCTTTTTTTCCTGTGGGGGACA1140    AGGTTCGCGTCCTCAACAAGAACCCGGTGGGCCATACCCGCATGCCGCGCTACACGCGGG1200    CAAAGTGGGGACAGTGGTCATCGACCATGGTGTGTTTCGTGACGCCGGACACCGCGGCAC1260    ACGGAAAGGGCGAGCAGCCCCAGCACGTTTACACCGTGAGTTTCACGTCGGTCGAACTGT1320    GGGGGCAAGACGCTTCCTCGCCGAAGGACACGATTCGCGTCGACTTGTGGGATGACTACC1380    TGGAGCCAGCGTGATCATGAAAGACGAACGGTTTCCATTGCCAGAGGGTTCGCTGAAGGA1440    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    GAGGAATTCATGGCCATTACTCGCCCTACCC31    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    GTCGAATTCTCAGAGCGTGCGCCAGTCCACC31    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1521 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    ATGGCCATTACTCGCCCTACCCTCGACCAGGTTTTAGACATCCGAACCCAGTTGCACATG60    CAACTGACGCACGAACAGGCAGCGTCCTACCTGGAACTGATGCAACCGAGTTTCGACGCC120    TACGACCTGGTCGACGAACTGGCTGATTTCGTTCCGCCAATACGCTACGACCGCAGTTCA180    GGCTATCGCCATCGGCCATCGGCCAAGGAAAACCCTCTGAACGCCTGGTACTACCGAACA240    GAAGTGAATGGTGCCCGCGAAGGCCTGCTGGCGGGCAAAACCGTCGCGCTCAAAGATAAT300    ATCTCCCTGGCAGGCGTCCCCATGATGAACGGCGCAGCGCCGTTGGAAGGCTTCGTCCCG360    GGGTTCGATGCCACGGTGGTCACCCGCTTGCTCGATGCGGGGGCGACCATTCTCGGCAAA420    GCCACCTGCGAGCACTACTGCCTTTCAGGAGGCAGCCACACCTCCGATCCAGCCCCGGTG480    CACAACCCACATCGCCACGGTTATGCCTCTGGCGGTTCCTCATCAGGCAGCGCGGCATTG540    GTTGCGTCCGGTGAGGTGGACATCGCCGTGGGCGGCGATCAAGGCGGCTCCATTCGGATC600    CCGTCGGCCTTCTGCGGTACCTACGGCATGAAGCCCACCCACGGCCTGGTGCCCTACACC660    GGCGTCATGGCGATTGAAGCCACGATCGATCATGTCGGCCCCATCACCGGTAACGTGCGC720    GACAACGCGCTGATGCTGCAGGCAATGGCCGGTGCAGACGGACTCGACCCGCGCCAGGCG780    GCGCCTCAGGTCGATGACTATTGCAGTTACCTGGAAAAAGGCGTGAGCGGACTCAGAATC840    GGGGTGTTGCAAGAGGGATTCGCGCTTGCTAACCAGGACCCTCGCGTGGCGGACAAAGTG900    CGCGACGCCATCGCCCGACTCGAGGCGTTGGGCGCTCATGTCGAGCCGGTCTCCATTCCC960    GAGCACAACCTGGCAGGGTTGTTGTGGCACCCCATCGGTTGCGAAGGCTTGACCATGCAG1020    ATGATGCATGGCAACGGCGCAGGCTTTAACTGGAAAGGACTTTACGATGTCGGCCTGCTG1080    GACAAACAAGCCAGCTGGCGCGACGACGCAGACCAATTATCCGCGTCGCTCAAGCTCTGC1140    ATGTTCGTCGGCCAATACGGCCTGTCGCGCTACAACGGACGCTACTACGCCAAGGCCCAG1200    AACCTTGCACGCTTTGCCCGGCAGGGATACGACAAAGCGCTGCAAACCTATGACCTGCTG1260    GTGATGCCGACCACGCCCATCACGGCCCAACCCCACCCGCCAGCGAACTGCTCGATCACG1320    GAGTACGTGGCTCGCGCGTTGGAAATGATCGGCAATACCGCGCCACAGGACATCACCGGG1380    CATCCGGCCATGTCGATTCCGTGTGGCCTGCTGGACGGCCTGCCCGTCGGGCTGATGCTG1440    GTCGCAAAACACTACGCCGAGGGCACGATTTACCAAGCGGCGGCGGCGTTTGAAGCCTCG1500    GTGGACTGGCGCACGCTCTGA1521    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 384 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (B) STRAIN: P14K    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    ATGGCCCTGTGTTTGACGAGCCTTGGCAGTCCCAGGCGTTTGCCTTGGTGGTCAGCATGC60    ACAAGGCCGGTCTCTTTCAGTGGAAAGACTGGGCCGAGACCTTCACCGCCGAAATCGACG120    CTTCCCCGCTCTGCCGGCGAAAGCGTCAACGACACCTACTACCGGCAATGGGTGTCGGCG180    CTGGAAAAGTTGGTGGCGTCGCTGGGGCTTGTGACGGGTGGAGACGTCAACTCGCGCGCA240    CAGGAGTGGAAACAGGCCCACCTCAACACCCCACATGGGCACCCGATCCTGCTGGCCCAT300    GCGCTTTGCCCGCCAGCGATCGACCCCAAGCACAAGCACGAGCCACAACGCTCACCGATC360    AAGGTCGTTGCCGCAATGGCTTGA384    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 127 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (B) STRAIN: P14K    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    MetAlaLeuCysLeuThrSerLeuGlySerProArgArgLeuProTrp    151015    TrpSerAlaCysThrArgProValSerPheSerGlyLysThrGlyPro    202530    ArgProSerProProLysSerThrLeuProArgSerAlaGlyGluSer    354045    ValAsnAspThrTyrTyrArgGlnTrpValSerAlaLeuGluLysLeu    505560    ValAlaSerLeuGlyLeuValThrGlyGlyAspValAsnSerArgAla    65707580    GlnGluTrpLysGlnAlaHisLeuAsnThrProHisGlyHisProIle    859095    LeuLeuAlaHisAlaLeuCysProProAlaIleAspProLysHisLys    100105110    HisGluProGlnArgSerProIleLysValValAlaAlaMetAla    115120125    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    GATGCGGCCATAGGCGAATTC21    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    ACCGCCACCGACTACCTGCAG21    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:    GTCAGCCTGAGCAATCTGCAG21    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:    GAATTCGGAAAAAATCGTACG21    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1401 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (B) STRAIN: AMIDASE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:    ATGAGTTCGCTAACCCGCCTCACCCTCGCGCAAGTTGCGCAGAAACTTAAGGCACGGGAA60    GTCTCCGCCGTTGAAGTTCTGGACGCCTGTCTGACGCAGGTGCGCTCCACCGAAAAACAG120    ATCAGTGCGTACGTGTGCGTGCTGGAGGATCAGGCCCGTGCAGCAGCCCACGCAACTGAC180    GCCGACATCCGCGGGCGCTGGAAAGGCCCGCTGCATGGCGTGCCTGTAGCGGTCAAGGAC240    TTATACGACATCGCTGGCGTACCCACCACGGCATCGTCGCCAGCGCACGAATTGGACGCG300    CAGCAAGACCCGGCTAGAGTCCGGCGCTTACAAGACGCAGGTGCCGTTATCCTTGGCAAG360    ACCCATACGCACGAATTCGCCTATGGCCGCATCACTCCGAAGTCGCGCAACCCCAGGGAC420    CCGGGAAGAACACCGGGTGGCTCCAGCGGCGGCTCGGCGGCCACGGTCGCAGCCTGCTGC480    GTCTACTTGGCGACCGGCACCGACACCGGTGGATCCGTTCGCATCCCTTCGTCGATGTGC540    AACACCGTAGGCCTGAAGCAACCTACGGTCGGCCGCGTGCACGGTGCCGGTGTGAGTTCA600    CTTTCCTGGAGCCTGGACCATCCAGGCCCGATCACGCGCACCGTGGAAGACACGGCGCTC660    ATGCTTCAGGTGATGGCTGGCTTCGATCCAGCCGACCCGCGGTCGTTGGATGAGCCGGTG720    CCCAGCTATGCCGAAGGGCTCGGCCAAGGCGTGAAAGGCCTGCGCTGGGGTGTGCCGAAG780    AACTACTTCTTCGACCGCGTGGACCCGGAAGTTGAAAGTGCGGTTCGTGCCGCCATCGAT840    CAACTGAAAGAGCTGGGCGCCGAACTGGTGGAAGTCGAAGTGCCCATGGCCGAGCAGATC900    ATCCCGGTGAAGTTCGGGATCATGCTACCCGAAGCCAGCGCCTACCACCGCACGATGCTG960    CGCGAGTCACCCGAGCTCTACACCGCCGATGTCCGCATACTGCTGGAACTCGGAGATCTA1020    GTCACCGCCACCGACTACCTGCAGGCGCAGCGCGTCCGTACGCTGATGCAGCGCGCGGTG1080    GCCGAGATGTACCAGCGCATCGATGTGCTGATCGCACCCACACTGCCCATCCCGGCTGCT1140    CGCAGCGGGGAGGAGGTCCACACATGGCCGGACGGCACGGTAGAGGCGTTGGTCATGGCC1200    TATACGCGCTTCACCTCGTTCGGCAACGTGACAGGATTACCCACGCTGAACCTGCCCTGT1260    GGTTTCTCCAAGGATGGGTTGCGATCGGCATGCAGATCAGGCCGGCCGCTGGACGAGAAG1320    ACCCTGCTGCGTGCTGGGCTGGCCTACGAGAAAGCCACGACCTGGCACCAGCGTCATCCG1380    GAACTGATCGGAGCGGGCTGA1401    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 466 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (B) STRAIN: AMIDASE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:    MetSerSerLeuThrArgLeuThrLeuAlaGlnValAlaGlnLysLeu    151015    LysAlaArgGluValSerAlaValGluValLeuAspAlaCysLeuThr    202530    GlnValArgSerThrGluLysGlnIleSerAlaTyrValCysValLeu    354045    GluAspGlnAlaArgAlaAlaAlaHisAlaThrAspAlaAspIleArg    505560    GlyArgTrpLysGlyProLeuHisGlyValProValAlaValLysAsp    65707580    LeuTyrAspIleAlaGlyValProThrThrAlaSerSerProAlaHis    859095    GluLeuAspAlaGlnGlnAspProAlaArgValArgArgLeuGlnAsp    100105110    AlaGlyAlaValIleLeuGlyLysThrHisThrHisGluPheAlaTyr    115120125    GlyArgIleThrProLysSerArgAsnProArgAspProGlyArgThr    130135140    ProGlyGlySerSerGlyGlySerAlaAlaThrValAlaAlaCysCys    145150155160    ValTyrLeuAlaThrGlyThrAspThrGlyGlySerValArgIlePro    165170175    SerSerMetCysAsnThrValGlyLeuLysGlnProThrValGlyArg    180185190    ValHisGlyAlaGlyValSerSerLeuSerTrpSerLeuAspHisPro    195200205    GlyProIleThrArgThrValGluAspThrAlaLeuMetLeuGlnVal    210215220    MetAlaGlyPheAspProAlaAspProArgSerLeuAspGluProVal    225230235240    ProSerTyrAlaGluGlyLeuGlyGlnGlyValLysGlyLeuArgTrp    245250255    GlyValProLysAsnTyrPhePheAspArgValAspProGluValGlu    260265270    SerAlaValArgAlaAlaIleAspGlnLeuLysGluLeuGlyAlaGlu    275280285    LeuValGluValGluValProMetAlaGluGlnIleIleProValLys    290295300    PheGlyIleMetLeuProGluAlaSerAlaTyrHisArgThrMetLeu    305310315320    ArgGluSerProGluLeuTyrThrAlaAspValArgIleLeuLeuGlu    325330335    LeuGlyAspLeuValThrAlaThrAspTyrLeuGlnAlaGlnArgVal    340345350    ArgThrLeuMetGlnArgAlaValAlaGluMetTyrGlnArgIleAsp    355360365    ValLeuIleAlaProThrLeuProIleProAlaAlaArgSerGlyGlu    370375380    GluValHisThrTrpProAspGlyThrValGluAlaLeuValMetAla    385390395400    TyrThrArgPheThrSerPheGlyAsnValThrGlyLeuProThrLeu    405410415    AsnLeuProCysGlyPheSerLysAspGlyLeuArgSerAlaCysArg    420425430    SerGlyArgProLeuAspGluLysThrLeuLeuArgAlaGlyLeuAla    435440445    TyrGluLysAlaThrThrTrpHisGlnArgHisProGluLeuIleGly    450455460    AlaGly    465    __________________________________________________________________________

What is claimed is:
 1. A method for converting a nitrile of the formula##STR7## wherein: A is selected from the group consisting of: ##STR8##to the corresponding amide comprising 1) contacting said nitrile withthe transformed host cell comprising a nucleic acid fragment having thenucleotide sequence represented by SEQ ID NO.:17 that stereospecificallyconverts the racemic nitrile to the corresponding enantiomeric R- orS-amide, the host cell selected from the group consisting ofEscherichia, Pseudomonas, Rhodococcus, Acinetobacter, Bacillus,Streptomyces, Hansenula, Saccharomyces, Pichia, Aspergillus, Neurospora,and Penicillium,2) recovering the product of Step 1).
 2. A method forconverting a nitrile of the formula ##STR9## wherein: A is selected fromthe group consisting of: ##STR10## to the corresponding enantiomeric (R)or (S)-carboxylic acid comprising 1) contacting the nitrile with thetransformed host that comprises a 5.0 kb EcoR1/Hind3 fragment of a 6.5kb nucleic acid fragment as characterized by the restriction map shownin FIG. 2 and a nucleic acid fragment having the sequence of SEQ IDNO:20, wherein said host stereospecifically converts the racemic nitrileto the corresponding enantiomeric (R)- or (S)-carboxylic acid, the hostcell selected from the group consisting of Escherichia, Pseudomonas,Rhodococcus, Acinetobacter, Bacillus, Streptomyces, Hansenula,Saccharomyces, Pichia, Aspergillus, Neurospora, and Penicillium, and2)recovering the product of Step 1).
 3. A method for converting a nitrileof the formula ##STR11## wherein: A is selected from the groupconsisting of: ##STR12## to the corresponding amide comprising 1)contacting said nitrile with a transformed host cell comprising thenucleic acid fragment having the sequence of SEQ ID NO:17 and thenucleic acid frogment corresponding to the P14K region of a 6.5 kbnucleic acid fragment as shown in FIG. 10 wherein said fragment encodesa polypeptide having an aminp acis sequence of SEQ ID NO:22 the hostcell capable of stereospecifically converting the racemic nitrile to thecorresponding enantiomeric R- or S-amide, the host cell selected fromthe group consisting of Escherichia, Pseudomonas, Rhodococcus,Acinetobacter, Bacillus, Streptomyces, Hansenula, Saccharomyces, Pichia,Aspergillus, Neurospora, and Penicillium,2) recovering the product ofStep 1).
 4. A method for converting a nitrile of the formula ##STR13##wherein: A is selected from the group consisting of: ##STR14## to thecorresponding enantiomeric (R) or (S)-carboxylic acid comprising 1)contacting the nitrile with the transformed Pichia pastoris comprisingpSW50 and identified as ATCC 74391 that stereospecifically converts theracemic nitrile to the corresponding enantiomeric (R)- or (S)-carboxylicacid, the host cell selected from the group consisting of Escherichia,Pseudomonas, Rhodococcus, Acinetobacter, Bacillus, Streptomyces,Hansenula, Saccharomyces, Pichia, Aspergillus, Neurospora, andPenicillium, and2) recovering the product of Step 1).